The present invention is directed to methods and compositions that are effective to enhance transport of biologically active agents, such as organic compounds, polypeptides, oligosaccharides, nucleic acids, and metal ions, across biological membranes.
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The plasma membranes of cells present a barrier to passage of many useful therapeutic agents. In general, a drug must be freely soluble in both the aqueous compartments of the body and the lipid layers through which it must pass, in order to enter cells. Highly charged molecules in particular experience difficulty in passing across membranes. Many therapeutic macromolecules such as peptides and oligonucleotides are also particularly intractable to transmembrane transport. Thus, while biotechnology has made available a greater number of potentially valuable therapeutics, bioavailability considerations often hinder their medicinal utility. There is therefore a need for reliable means of transporting drugs, and particularly macromolecules, into cells.
Heretofore, a number of transporter molecules have been proposed to escort molecules across biological membranes. Ryser et al. (1979) teaches the use of high molecular weight polymers of lysine for increasing transport of various molecules across cellular membranes, with very high molecular weights being preferred. Although the authors contemplated polymers of other positively charged residues such as ornithine and arginine, operativity of such polymers was not shown.
Barsoum et al. (1994) and Fawell et al. (1994) proposed using shorter fragments of the tat protein containing the tat basic region (residues 49-57 having the sequence RKKRRQRRR (SEQ ID NO: 1). Barsoum et al. noted that moderately long polyarginine polymers (MW 5000-15000 datons) failed to enable transport of .beta.-galactosidase across cell membranes (e.g., Barsourm on page 3), contrary to the suggestion of Ryser et al. (supra).
Other studies have shown that a 16 amino acid peptide-cholesterol conjugate derived from the Antennapedia homeodomain is rapidly internalized by cultured neurons (Brugidou et al., 1995). However, slightly shorter versions of this peptide (is residues) are not effectively taken up by cells (Derossi et al., 1996).
The present invention is based in part on the applicants"" discovery that conjugation of certain polymers composed of contiguous, highly basic subunits, particularly subunits containing guanidyl or amidinyl moieties, to small molecules or macromolecules is effective to significantly enhance transport of the attached molecule across biological membranes. Moreover, transport occurs at a rate significantly greater than the transport rate provided by a basic HIV tat peptide consisting of residues 49-57 (SEQ ID NO: 1).
The present invention includes, in one aspect, a method for enhancing transport of a selected compound across a biological membrane. In the method, a biological membrane is contacted with a conjugate containing a biologically active agent that is covalently attached to at least one transport polymer. The conjugate is effective to promote transport of the agent across the biological membrane at a rate that is greater than the trans-membrane transport rate of the biological agent in non-conjugated form.
In one embodiment, the polymer consists of from 6 to 25 subunits, at least 50% of which contain a guanidino or amidino sidechain moiety, wherein the polymer contains at least 6, and more preferably, at least 7 guanidino or amidino sidechain moieties. In another embodiment, the polymer consists of from 6 to 20, 7 to 20, or 7 to 15 subunits. More preferably, at least 70% of the subunits in the polymer contain guanidino or amidino sidechain moiety, and more preferably still, 90%. Preferably, no guanidino or amidino sidechain moiety is separated from another such moiety by more than one non-guanidino or non-amidino subunit. In a more specific embodiment, the polymer contains at least 6 contiguous subunits each containing either a guanidino or amidino group, and preferably at least 6 or 7 contiguous guanidino sidechain moieties.
In another embodiment, the transport polymer contains from 6 to 25 contiguous subunits, from 7 to 25, from 6 to 20, or preferably from 7 to 20 contiguous subunits, each of which contains a guanidino or amidino sidechain moiety, and with the optional proviso that one of the contiguous subunits can contain a non-arginine residue to which the agent is attached.
In one embodiment, each contiguous subunit contains a guanidino moiety, as exemplified by a polymer containing at least six contiguous arginine residues.
Preferably, each transport polymer is linear. In a preferred embodiment, the agent is attached to a terminal end of the transport polymer.
In another specific embodiment, the conjugate contains a single transport polymer.
The transport-enhancing polymers are exemplified, in a preferred embodiment, by peptides in which arginine residues constitute the subunits. Such a polyarginine peptide may be composed of either all D-, all L- or mixed D- and L-arginines, and may include additional amino acids. More preferably, at least one, and preferably all of the subunits are D-arginine residues, to enhance biological stability of the polymer during transit of the conjugate to its biological target.
The method may be used to enhance transport of selected therapeutic agents across any of a number of biological membranes including, but not limited to, eukaryotic cell membranes, prokaryotic cell membranes, and cell walls. Exemplary prokaryotic cell membranes include bacterial membranes. Exemplary eukaryotic cell membranes of interest include, but are not limited to membranes of dendritic cells, epithelial cells, endothelial cells, keratinocytes, muscle cells, fungal cells, bacterial cells, plant cells, and the like.
According to a preferred embodiment of the invention, the transport polymer of the invention has an apparent affinity (Km) that is at least 10-fold greater, and preferably at least 100-fold greater, than the affinity measured for tat(49-75) peptide by the procedure of Example 6 when measured at room temperature (23xc2x0 C.) or 37xc2x0 C.
Biologically active agents (which encompass therapeutic agents) include, but are not limited to metal ions, which are typically delivered as metal chelates; small organic molecules, such anticancer (e.g., taxane) and antimicrobial molecules (e.g., against bacteria or fungi such as yeast); and macromolecules such as nucleic acids, peptides, proteins, and analogs thereof. In one preferred embodiment, the agent is a nucleic acid or nucleic acid analog, such as a ribozyme which optionally contains one or more 2xe2x80x2-deoxy nucleotide subunits for enhanced stability. Alternatively, the agent is a peptide nucleic acid (PNA). In another preferred embodiment, the agent is a polypeptide, such as a protein antigen, and the biological membrane is a cell membrane of an antigen-presenting cell (APC). In another embodiment, the agent is selected to promote or elicit an immune response against a selected tumor antigen. In another preferred embodiment, the agent is a taxane or taxoid anticancer compound. In another embodiment, the agent is a non-polypeptide agent, preferably a non-polypeptide therapeutic agent. In a more general embodiment, the agent preferably has a molecular weight less than 10 kDa.
The agent may be linked to the polymer by a linking moiety, which may impart conformational flexibility within the conjugate and facilitate interactions between the agent and its biological target. In one embodiment, the linking moiety is a cleavable linker, e.g., containing a linker group that is cleavable by an enzyme or by solvent-mediated cleavage, such as an ester, amide, or disulfide group. In another embodiment, the cleavable linker contains a photocleavable group.
In a more specific embodiment, the cleavable linker contains a first cleavable group that is distal to the biologically active agent, and a second cleavable group that is proximal to the agent, such that cleavage of the first cleavable group yields a linker-agent conjugate containing a nucleophilic moiety capable of reacting intramolecularly to cleave the second cleavable group, thereby releasing the agent from the linker and polymer.
In another embodiment, the invention can be used to screen a plurality of conjugates for a selected biological activity, wherein the conjugates are formed from a plurality of candidate agents. The conjugates are contacted with a cell that exhibits a detectable signal upon uptake of the conjugate into the cell, such that the magnitude of the signal is indicative of the efficacy of the conjugate with respect to the selected biological activity. This method is particularly useful for testing the activities of agents that by themselves are unable, or poorly able, to enter cells to manifest biological activity. In one embodiment, the candidate agents are selected from a combinatorial library.
The invention also includes a conjugate library which is useful for screening in the above method.
In another aspect, the invention includes a pharmaceutical composition for delivering a biologically active agent across a biological membrane. The composition comprises a conjugate containing a biologically active agent covalently attached to at least one transport polymer as described above, and a pharmaceutically acceptable excipient. The polymer is effective to impart to the agent a rate of trans-membrane transport that is greater than the trans-membrane transport rate of the agent in non-conjugated form. The composition may additionally be packaged with instructions for using it.
In another aspect, the invention includes a therapeutic method for treating a mammalian subject, particularly a human subject, with a pharmaceutical composition as above.
These and other objects and features of the invention will become more fully apparent when the following detailed description of the invention is read in conjunction with the accompanying drawings.